The invention relates generally to the field of molecular biology. Certain embodiments entail materials and methods suitable for the biosynthesis of biopolymers, namely polyhydroxyalkanoic acid polymers.
The production of intracellular polyesters belonging to the class of polymers known as polyhydroxyalkanoates (polyhydroxyalkanoic acids) has been observed in a wide array of prokaryotic organisms (Anderson, A. J. and Dawes, E. A. (1990) Microbiol. Rev. 54:450-472; Steinbxc3xcchel, A. and Valentin, H. E. (1995) FEMS Microbiol. Lett. 128:219-228). The monomers composing the polyesters range in length from C4 (3-hydroxybutyrate) to C12 (3-hydroxydodecanoate) (Lageveen, R. G. et al. (1988) Appl. Env. Microbiol. 54:2924-2932). These polyesters have attracted considerable interest as they are biodegradable. Potential technical applications exist in industry and agriculture, as well as in medical devices and procedures (Hocking, P. J. and Marchessault, R. H. (1994) Biopolyesters. In: G. J. L. Griffin (Ed) Chemistry and technology of biodegradable polymers, Chapman and Hall, London, pp.48-96; Mxc3xcller, H. M. and Seebach, D. (1993) Angew. Chem. 105:483-509). Additionally, this class of polyesters is attractive as a potential alternative to conventional petrochemical-derived plastics.
Polyhydroxyalkanoic acids are broadly characterized according to the monomers that constitute their backbone. Polymers composed of C4-C5 units are classified as short chain length (scl) polyhydroxyalkanoic acids; polymers containing monomers of C6 units and above are classified as medium chain length (mcl) polyhydroxyalkanoic acids. The primary structure of the polymer influences the physical properties of the polyester.
The metabolic pathways leading to the formation of polyhydroxyalkanoic acids have not been elucidated for all organisms. The most extensively studied polyhydroxyalkanoic acid biosynthetic pathway is that of Alcaligenes (Peoples, O. P. et al. (1989) J. Biol. Chem. 264:15298-15303; Valentin, H. E. et al. (1995) Eur. J. Biochem. 227:43-60). This organism is capable of forming either a homopolymer of C4 (polyhydroxybutyrate, PHB) or a co-polymer of C4-C5 (PHB-PHV, polyhydroxybutyrate-polyhydroxyvalerate) (Koyama, N. and Doi, Y. (1995) Biotechnol. Lett. 17:281-284). Hence, A. eutrophus is classified as a scl polyhydroxyalkanoic acid organism. Similarly, Pseudomonas species generate a polymer composed of monomers ranging in length from C6 to C12 (Timm, A. and Steinbxc3xcchel, A. (1990) Appl. Environ. Microbiol. 56:3360; Lageveen, R. G. et al. (1988) Appl. Env. Microbiol. 54:2924-2932), and are classified as mcl polyhydroxyalkanoic acid organisms.
The polymerization of the hydroxyacyl-CoA substrates is carried out by polyhydroxyalkanoic acid synthases. The substrate specificity of this class of enzyme varies across the spectrum of polyhydroxyalkanoic acid producing organisms. This variation in substrate specificity of polyhydroxyalkanoic acid synthases is supported by indirect evidence observed in heterologous expression studies (Lee, E. Y. et al. (1995) Appl. Microbiol. Biotechnol. 42:901-909; Timm, A. et al. (1990) Appl. Microbiol. Biotechnol. 33:296-301). Hence, the structure of the backbone of the polymer is strongly influenced by the polyhydroxyalkanoic acid synthase responsible for its formation.
Fluorescent pseudomonads belonging to the rRNA homology group I can synthesize and accumulate large amounts of polyhydroxyalkanoic acids (PHA) composed of various saturated and unsaturated hydroxy fatty acids with carbon chain lengths ranging from 6 to 14 carbon atoms (Steinbxc3xcchel, A. and Valentin, H. E. (1992) FEMS Microbiol. Rev. 103:217). Polyhydroxyalkanoic acid isolated from these bacteria also contains constituents with functional groups such as branched, halogenated, aromatic or nitrile side-chains (Steinbxc3xcchel and Valentin (1995 FEMS Microbiol. Lett. 128:219-228). The composition of polyhydroxyalkanoic acid depends on the polyhydroxyalkanoic acid polymerase system the carbon source, and the metabolic routes (Anderson, A. J. and Dawes, E. A. (1990) Microbiol. Rev. 54:450-472; Eggink et al. (1992) FEMS Microbiol. Rev. 105:759; Huisman, A. M. et al. (1989) Appl. Microbiol. Biotechnol. 55:1949-1954; Lenz, O. et al. (1992) J. Bacteriol. 176:4385-4393; Steinbxc3xcchel, A. and Valentin, H. E. (1995) FEMS Microbiol. Lett. 128:219-228). In P. putida, at least three different metabolic routes occur for the synthesis of 3-hydroxyacyl CoA thioesters, which are the substrates of the polyhydroxyalkanoic acid synthase (Huijberts, G. N. M. et al. (1994) J. Bacteriol. 176:1661-1666): (i) xcex2-oxidation is the main pathway when fatty acids are used as carbon source; (ii) De novo fatty acid biosynthesis is the main route during growth on carbon sources which are metabolized to acetyl-CoA, like gluconate, acetate or ethanol; and (iii) Chain elongation reaction, in which acyl-CoA is condensed with acetyl-CoA to the two carbon chain extended xcex2-keto product which is then reduced to 3-hydroxyacyl-CoA. This latter pathway is involved in polyhydroxyalkanoic acid-synthesis during growth on hexanoate.
The polyhydroxyalkanoic acid synthase structural gene from Alcaligenes eutrophus (phaCAe) has been cloned and characterized at the molecular level in several laboratories (for a review see Steinbxc3xcchel, A. and Schlegel, H. G. (1991) Mol. Microbiol. 5:535-542; GenBank Accession number J05003). It was demonstrated that phaCAe in combination with other genes conferred the ability to synthesize poly(3-hydroxybutyric acid) not only to many bacteria, which do not synthesize this polyester such as e.g. Escherichia coli (Steinbxc3xcchel, A. and Schlegel, H. G. (1991) Mol. Microbiol. 5:535-542) but also to Saccharomyces cerevisiae (Leaf, T. A. et al. (1996) Microbiology 142:1169-1180), plants such as Arabidopsis thaliana (Poirier, Y. et al. (1992) Science 256:520-523.) and Gossypium hirsutum (John, M. E. and Keller, G. (1996) Proc. Natl. Acad. Sci. U.S.A. 93:12768-12773), and even to cells from the insect Spodoptera frugiperda (Williams, M. D. et al. (1996) Appl. Environ. Microbiol. 62:2540-2546).
The development of biological systems that synthesize poly(4-hydroxybutyric acid), poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid), and other polyester materials would be of great utility. Biological systems provide the potential to produce significant quantities of important materials, while utilizing inexpensive feedstocks and minimizing hazardous byproducts.
There exists a need for novel biosynthetic routes to polymers of potential commercial interest that do not rely on petroleum based starting materials Biological processes present an attractive alternative to chemical processes that produce potentially harmful byproducts while consuming non-renewable resources.
This invention relates to materials and processes for preparing polyester materials. More particularly, the invention is related to materials and processes for the preparation of polyester materials, preferably poly(4-hydroxybutyric acid), poly(3-hydroxybutyric acid), and poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid). The invention provides nucleic acid segments encoding a polyhydroxyalkanoic acid synthase protein and either a fatty acid:acyl-CoA transferase protein or an acyl-CoA synthetase protein, recombinant vectors, cells containing the nucleic acid segments, and methods for the preparation of polyester materials.
The scope of the present invention will be further apparent in light of the detailed descriptions provided below. However, it should be understood that the following detailed description and examples, while indicating preferred embodiments of the present invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the present invention will become apparent to those of ordinary skill in the art from this detailed description.
Definitions
The following definitions are provided in order to aid those skilled in the art in understanding the detailed description of the present invention.
An xe2x80x9cacyl-CoA synthetasexe2x80x9d or xe2x80x9cthiokinasexe2x80x9d protein catalyzes the formation of a thioester linkage between the carboxyl group of a fatty acid and the sulfhydryl group of CoA.
An xe2x80x9cacyl kinasexe2x80x9d protein catalyzes the transfer of a phosphate group from ATP to a carboxylate group according to the reaction: 
wherein R is an alkyl or hydroxyalkyl group.
xe2x80x9cCoAxe2x80x9d refers to coenzyme A.
The term xe2x80x9cfatty acid:acyl-Co A transferasexe2x80x9d refers to a protein that catalyzes an acyl group transfer according to the reaction: 
wherein R1 and R2 are alkyl or hydroxyalkyl groups. Groups R1 and R2 may further contain one or more double bonds, triple bonds, or aromatic groups.
xe2x80x9cCopolyesterxe2x80x9d refers to a polyester material made from a two different monomeric building blocks. For example, poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) is a polyester made from 3-hydroxybutyric acid and 4-hydroxybutyric acid. The relative composition of the two monomeric building blocks in the copolyester can be variable. Copolyesters are commonly characterized by the relative percentages of the two monomeric building blocks. The percentage composition may affect the physical characteristics of the copolyester.
xe2x80x9cDimericxe2x80x9d refers to enzymes that are comprised of two protein molecule subunits. The two subunits may be identical (homodimeric) or different (heterodimeric) in sequence.
xe2x80x9cHeterologousxe2x80x9d refers to nucleotide segments not normally found in nature in the same organism.
xe2x80x9cHomopolyesterxe2x80x9d refers to a polyester material made from a single monomeric building block. For example, poly(4-hydroxybutyric acid) is a polyester made from 4-hydroxybutyric acid.
A xe2x80x9c4-hydroxybutyrate dehydrogenasexe2x80x9d protein catalyzes the conversion of succinate semialdehyde to 4-hydroxybutyrate.
The combination of xe2x80x9c2-methylcitrate dehydratasexe2x80x9d protein and xe2x80x9c2-methylisocitrate dehydratasexe2x80x9d protein catalyzes the conversion of 2-methylcitrate to 2-methylisocitrate.
A xe2x80x9c2-methylcitrate synthasexe2x80x9d protein catalyzes the conversion of propionyl-CoA and oxaloacetate to 2-methylcitrate.
A xe2x80x9c2-methylisocitrate lyasexe2x80x9d protein catalyzes the conversion of 2-methylisocitrate to pyruvate and succinate.
The terms xe2x80x9cmicrobexe2x80x9d, xe2x80x9cmicroorganismxe2x80x9d, and xe2x80x9cmicrobialxe2x80x9d refer to algae, bacteria, fungi, and protozoa.
xe2x80x9cMonomericxe2x80x9d refers to enzymes that are comprised of a single protein molecule.
xe2x80x9cNativexe2x80x9d refers to two segments of nucleic acid naturally occurring in the same organism. For example, a native promoter is the promoter naturally found with a given gene in an organism.
xe2x80x9cNucleic acidxe2x80x9d refers to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
xe2x80x9cOverexpressionxe2x80x9d refers to the expression of a polypeptide or protein encoded by a DNA introduced into a host cell, wherein said polypeptide or protein is either not normally present in the host cell, or wherein said polypeptide or protein is present in said host cell at a higher level than that normally expressed from the endogenous gene encoding said polypeptide or protein.
A xe2x80x9c2-oxogluatarate decarboxylasexe2x80x9d protein catalyzes the conversion of 2-oxoglutarate to succinate semialdehyde.
A xe2x80x9cphosphotransacylasexe2x80x9d protein catalyzes the transfer of a phosphorylated acyl group to CoA according to the reaction: 
The phrases xe2x80x9cpolyhydroxyalkanoic acid biosynthetic genesxe2x80x9d and xe2x80x9cpolyhydroxyalkanoic acid biosynthetic enzymesxe2x80x9d refer to those genes or enzymes leading to anabolic reactions in the pathway of polyhydroxyalkanoic acid production.
The phrase xe2x80x9cpolyhydroxyalkanoate (PHA) synthasexe2x80x9d refers to enzymes that convert hydroxyacyl-CoAs to polyhydroxyalkanoates and free CoA.
The term xe2x80x9cpromoterxe2x80x9d or xe2x80x9cpromoter functional in bacteriaxe2x80x9d refer to a nucleotide sequence, usually found upstream (5xe2x80x2) to a coding sequence, that controls expression of the coding sequence by controlling production of messenger RNA (mRNA) by providing the recognition site for RNA polymerase and/or other factors necessary for the start of transcription at the correct site. The promoters disclosed herein, and biologically functional equivalents thereof, are responsible for driving the transcription of coding sequences under their control when introduced into a bacterial cell, as demonstrated by their ability to produce mRNA.
The terms xe2x80x9crecombinant vectorxe2x80x9d and xe2x80x9cvectorxe2x80x9d refer to any agent such as a plasmid, cosmid, virus, autonomously replicating sequence, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleotide sequence, derived from any source, capable of genomic integration or autonomous replication, comprising a DNA molecule in which one or more DNA sequences have been linked in a functionally operative manner (xe2x80x9coperatively linkedxe2x80x9d). Such recombinant DNA constructs or vectors are capable of introducing a 5xe2x80x2 regulatory sequence or promoter region and a DNA sequence for a selected gene product into a cell in such a manner that the DNA sequence is transcribed into a functional mRNA which is translated and therefore expressed.
A xe2x80x9csuccinate-semialdehyde dehydrogenasexe2x80x9d protein catalyzes the conversion of succinyl-CoA to succinate semialdehyde.
A xe2x80x9csuccinate:acetyl-CoA transferasexe2x80x9d protein catalyzes the conversion of succinate to succinyl-CoA.
The present invention provides a novel method for the preparation of polyester materials. In one important embodiment, co-expression of a polyhydroxyalkanoic acid synthase gene and a fatty acid:acyl-CoA transferase gene in a cell enable the biosynthesis of poly(4-hydroxybutyric acid). In an alternative embodiment, the co-expression of a polyhydroxyalkanoic acid synthase gene and a fatty acid:acyl-CoA transferase gene in bacteria leads to the biosynthesis of poly(3-hydroxybutyric acid). In a further alternative embodiment, co-expression of a polyhydroxyalkanoic acid synthase gene and a fatty acid:acyl-CoA transferase gene in a cell enable the biosynthesis of the copolyester poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid). In an alternative embodiment, co-expression of a polyhydroxyalkanoic acid synthase gene and a fatty acid:acyl-CoA transferase gene in a cell enable the biosynthesis of poly(4-hydroxybutyric acid).
Alternatively, the co-expression of a polyhydroxyalkanoic acid synthase gene and an acyl-CoA synthetase gene in a cell leads to the biosynthesis of poly(3-hydroxybutyric acid). In a further alternative embodiment, co-expression of a polyhydroxyalkanoic acid synthase gene and an acyl-CoA synthetase gene in a cell enable the biosynthesis of the copolyester poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid). In an alternative embodiment, co-expression of a polyhydroxyalkanoic acid synthase gene and an acyl-CoA synthetase gene in a cell enable the biosynthesis of poly(4-hydroxybutyric acid).
In one important embodiment, the invention provides a nucleic acid segment that encodes a polyhydroxyalkanoic acid synthase protein, and that encodes a fatty acid:acyl-CoA transferase protein. The polyhydroxyalkanoic acid synthase protein may generally be any polyhydroxyalkanoic acid synthase protein suitable for the production of polyester materials according to the inventive processes. The polyhydroxyalkanoic acid synthase protein may be monomeric or dimeric. Monomeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Rhizobium meliloti, Alcaligenes eutrophus, Alcaligenes sp., Rhizobium etli, Paracoccus denitrificans, Acinetobacter sp., Rhodobacter sphaeroides, Meihylobacterium extorquens, Pseudomonas oleovorans, Pseudomonas aeruginosa, Rhodococcus ruber, and Zoogloea ramigera. Dimeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Thiocystis violacea and Chromatium vinosum. Preferably, the polyhydroxyalkanoic acid synthase protein is an Alcaligenes euirophus polyhydroxyalkanoic acid synthase protein, and more preferably, the Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein encoded by the Alcaligenes eutrophus phaC polyhydroxyalkanoic acid synthase structural gene. The fatty acid:acyl-CoA transferase protein may be any fatty acid:acyl-CoA transferase protein suitable for the preparation of polyester materials according to the inventive processes. Fatty acid:acyl-CoA transferase proteins include, but are not limited to, 4-hydroxybutyrate:acetyl-CoA transferase from Clostridium aminobutyricum, propionate:acyl-CoA transferase from Clostridium propionicum, and succinate:acyl-CoA transferase from Clostridium kluyveri. Preferably, the fatty acid:acyl-CoA transferase protein is a 4-hydroxybutyrate:acyl-CoA transferase protein, more preferably a Clostridium kluyveri 4-hydroxybutyrate:acyl-CoA transferase protein, and most preferably, the Clostridium kluyveri 4-hydroxybutyrate:acyl-CoA transferase protein encoded by the Clostridium kluyveri orfZ 4-hydroxybutyrate:acyl-CoA transferase structural gene. The nucleic acid segment may comprise deoxyribonucleic acids (i.e. DNA) or ribonucleic acids (i.e. RNA). The nucleic acid segment may further be single or double stranded. The nucleic acid segment may further be linear or circular in conformation. The nucleic acid segment may further comprise a promoter sequence functional in bacterial cells. The promoter may be inducible or constitutive. Promoters functional in bacterial cells are generally known to those of skill in the art, and include, but are not limited to the lac promoter, the bla promoter, the PL promoter, the Ptrc promoter, and the T7 promoter.
In an alternative embodiment, the invention provides a nucleic acid segment that encodes a polyhydroxyalkanoic acid synthase protein, and that encodes an acyl-CoA synthetase protein. The polyhydroxyalkanoic acid synthase protein may generally be any polyhydroxyalkanoic acid synthase protein suitable for the production of polyester materials according to the inventive processes. The polyhydroxyalkanoic acid synthase protein may be monomeric or dimeric. Monomeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Rhizobium meliloti, Alcaligenes eutrophus, Alcaligenes sp., Rhizobium etli, Paracoccus denitrificans, Acinetobacter sp., Rhodobacter sphaeroides, Methylobacterium extorquens, Pseudomonas oleovorans, Pseudomonas aeruginosa, Rhodococcus ruber, and Zoogloea ramigera. Dimeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Thiocystis violacea and Chromatium vinosum. Preferably, the polyhydroxyalkanoic acid synthase protein is an Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein, and more preferably, the Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein encoded by the Alcaligenes eutrophus phaC polyhydroxyalkanoic acid synthase structural gene. The acyl-CoA synthetase protein may be any acyl-CoA synthetase protein suitable for the preparation of polyester materials according to the inventive processes. Sources of acyl-CoA synthetase proteins include, but are not limited to, Alcaligenes eutrophus, Methanothrix soehngenii, and Aspergillus nidulans. Preferably, the acyl-CoA synthetase protein is a thiokinase protein, and more preferably, a 4-hydroxybutyrate thiokinase protein. The nucleic acid segment may comprise deoxyribonucleic acids (i.e. DNA) or ribonucleic acids (i.e. RNA). The nucleic acid segment may further be single or double stranded. The nucleic acid segment may further be linear or circular in conformation. The nucleic acid segment may further comprise a promoter sequence functional in bacterial cells. The promoter may be inducible or constitutive. Promoters functional in bacterial cells are generally known to those of skill in the art, and include, but are not limited to the lac promoter, the bla promoter, the PL promoter, the Ptrc promoter, and the T7 promoter.
The invention further provides recombinant vectors comprising a nucleic acid segment, wherein the segment encodes a polyhydroxyalkanoic acid synthase protein and encodes a fatty acid:acyl-CoA transferase protein. This vector may generally be any vector suitable for the delivery of the nucleic acid into a cell. Vectors are well known to those of skill in the art, and include, but are not limited to, plasmids, artificial chromosomes, viruses, bacteriophage, cosmids, and phagemids. In a preferred embodiment, the vector may be pKSSE5.3 or pSKSE5.3 as disclosed herein.
In a preferred embodiment, the invention encompasses a cell comprising a nucleic acid segment encoding a polyhydroxyalkanoic acid synthase protein and encoding a fatty acid:acyl-CoA transferase protein. The polyhydroxyalkanoic acid synthase protein may generally be any polyhydroxyalkanoic acid synthase protein suitable for the production of polyester materials according to the inventive processes. The polyhydroxyalkanoic acid synthase protein may be monomeric or dimeric. Monomeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Rhizobium meliloti, Alcaligenes eutrophus, Alcaligenes sp., Rhizobium etli, Paracoccus denitrificans, Acinetobacter sp., Rhodobacter sphaeroides, Methylobacterium extorquens, Pseudomonas oleovorans, Pseudomonas aeruginosa, Rhodococcus ruber, and Zoogloea ramigera. Dimeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Thiocystis violacea and Chromatium vinosum. Preferably, the polyhydroxyalkanoic acid synthase protein is an Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein, and more preferably, the Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein encoded by the Alcaligenes eutrophus phaC polyhydroxyalkanoic acid synthase structural gene. The fatty acid:acyl-CoA transferase protein may be any fatty acid:acyl-CoA transferase protein suitable for the preparation of polyester materials according to the inventive processes. Fatty acid:acyl-CoA transferase proteins include, but are not limited to, 4-hydroxybutyrate:acyl-CoA transferase from Clostridium aminobutyricum, propionate:acyl-CoA transferase from Clostridium propionicum, and succinate:acyl-CoA transferase from Clostridium kluyveri. Preferably, the fatty acid:acyl-CoA transferase protein is a 4-hydroxybutyrate:acyl-CoA transferase protein, more preferably a Clostridium kluyveri 4-hydroxybutyrate:acyl-CoA transferase protein, and most preferably, the Clostridium kluyveri 4-hydroxybutyrate:acyl-CoA transferase protein encoded by the Clostridium kluyveri orfZ 4-hydroxybutyrate:acyl-CoA transferase structural gene. The cell may generally be any cell suitable for the preparation of polyester materials. The cell may be, but is not limited to, a plant cell, a mammalian cells, an insect cell, a fungal cell, and a bacterial cell. Preferably, the cell is a plant cell. Preferably, the bacterial cell is Escherichia coli, and more preferably, the bacterial cell is Escherichia coli strain XL1-Blue.
In an alternative embodiment, the invention encompasses a cell comprising a nucleic acid segment encoding a polyhydroxyalkanoic acid synthase protein and encoding an acyl-CoA synthetase protein. The polyhydroxyalkanoic acid synthase protein may generally be any polyhydroxyalkanoic acid synthase protein suitable for the production of polyester materials according to the inventive processes. The polyhydroxyalkanoic acid synthase protein may be monomeric or dimeric. Monomeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Rhizobium meliloti, Alcaligenes eutrophus, Alcaligenes sp., Rhizobium etli, Paracoccus denitrificans, Acinetobacter sp., Rhodobacter sphaeroides, Methylobacterium extorquens, Pseudomonas oleovorans, Pseudomonas aeruginosa, Rhodococcus ruber, and Zoogloea ramigera. Dimeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Thiocystis violacea and Chromatium vinosum. Preferably, the polyhydroxyalkanoic acid synthase protein is an Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein, and more preferably, the Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein encoded by the Alcaligenes eutrophus phaC polyhydroxyalkanoic acid synthase structural gene. The acyl-CoA synthetase protein may be any acyl-CoA synthetase protein suitable for the preparation of polyester materials according to the inventive processes. Sources of acyl-CoA synthetase proteins include, but are not limited to, Alcaligenes eutrophus, Methanothrix soehngenii, and Aspergillus nidulans. Preferably, the acyl-CoA synthetase protein is a thiokinase protein, and more preferably a 4-hydroxybutyrate thiokinase protein. The cell may generally be any cell suitable for the preparation of polyester materials. The cell may be, but is not limited to, a plant cell, a mammalian cells, an insect cell, a fungal cell, and a bacterial cell. Preferably, the cell is a plant cell. Preferably, the bacterial cell is Escherichia coli, and more preferably, the bacterial cell is Escherichia coli strain XL1-Blue.
The invention further discloses methods for the preparation of a transformed cell, the transformed cell being suitable for the preparation of polyester materials. The type of cell includes, but is not limited to, a plant cell, a mammalian cell, an insect cell, a fungal cell, and a bacterial cell. Preferably, the cell is a plant cell. Alternatively, the cell is preferably a bacterial cell, more preferably the bacterial cell is Escherichia coli, and most preferably, the bacterial cell is Escherichia coli strain XL1-Blue. Means for the transformation of plants are well known in the art. Methods include, but are not limited to, liposome mediated transformation, electroporation, treatment with chemicals that increase free DNA uptake, free DNA delivery via microparticle bombardment, and transformation using viruses or pollen. Means for the transformation of bacterial cells are also well known in the art. Methods include, but are not limited to, electroporation, calcium chloride mediated transformation, and polyethylene glycol mediated transformation. The disclosed transformation methods comprise selecting a host cell, contacting the host cell with a nucleic acid segment encoding a polyhydroxyalkanoic acid synthase protein and encoding a fatty acid:acyl-CoA transferase protein, the contacting step being performed under conditions suitable for uptake of the nucleic acid segment by the cell. Subsequent regeneration of the cell affords the transformed cell. The polyhydroxyalkanoic acid synthase protein may generally be any polyhydroxyalkanoic acid synthase protein suitable for the production of polyester materials according to the inventive processes. The polyhydroxyalkanoic acid synthase protein may be monomeric or dimeric. Monomeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Rhizobium meliloti, Alcaligenes eutrophus, Alcaligenes sp., Rhizobium etli, Paracoccus denitrificans, Acinetobacter sp., Rhodobacter sphaeroides, Methylobacterium extorquens, Pseudomonas oleovorans, Pseudomonas aeruginosa, Rhodococcus ruber, and Zoogloea ramigera. Dimeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Thiocystis violacea and Chromatium vinosum. Preferably, the polyhydroxyalkanoic acid synthase protein is an Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein, and more preferably, the Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein encoded by the Alcaligenes eutrophus phaC polyhydroxyalkanoic acid synthase structural gene. The fatty acid:acyl-CoA transferase protein may be any fatty acid:acyl-CoA transferase protein suitable for the preparation of polyester materials according to the inventive processes. Fatty acid:acyl-CoA transferase proteins include, but are not limited to, 4-hydroxybutyrate:acetyl-CoA acyltransferase from Clostridium aminobutyricum, propionate:acetyl-CoA acyltransferase from Clostridium propionicum, and succinate:acetyl-CoA transferase from Clostridium kluyveri. Preferably, the fatty acid:acyl-CoA transferase protein is a 4-hydroxybutyrate:acyl-CoA transferase protein, more preferably a Clostridium kluyveri 4-hydroxybutyrate:acyl-CoA transferase protein, and most preferably, the Clostridium kluyveri 4-hydroxybutyrate:acyl-CoA transferase protein encoded by the Clostridium kluyveri orfZ 4-hydroxybutyrate:acyl-CoA transferase structural gene.
The invention discloses alternative methods for the preparation of a transformed cell, the transformed cell being suitable for the preparation of polyester materials. The disclosed transformation methods comprise selecting a host cell, contacting the host cell with a nucleic acid segment encoding a polyhydroxyalkanoic acid synthase protein and encoding an acyl-CoA synthetase protein, the contacting step being performed under conditions suitable for uptake of the nucleic acid segment by the cell. Subsequent regeneration of the cell affords the transformed cell. The type of cell includes, but is not limited to, a plant cell, a mammalian cell, an insect cell, a fungal cell, and a bacterial cell. Preferably, the cell is a plant cell. Alternatively, the cell is preferably a bacterial cell, more preferably the bacterial cell is Escherichia coli, and most preferably, the bacterial cell is Escherichia coli strain XL1-Blue. Means for the transformation of plants are well known in the art. Methods include, but are not limited to, liposome mediated transformation, electroporation, treatment with chemicals that increase free DNA uptake, free DNA delivery via microparticle bombardment, and transformation using viruses or pollen. Means for the transformation of bacterial cells are also well known in the art. Methods include, but are not limited to, electroporation, calcium chloride mediated transformation, and polyethylene glycol mediated transformation. The polyhydroxyalkanoic acid synthase protein may generally be any polyhydroxyalkanoic acid synthase protein suitable for the production of polyester materials according to the inventive processes. The polyhydroxyalkanoic acid synthase protein may be monomeric or dimeric. Monomeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Rhizobium meliloti, Alcaligenes eutrophus, Alcaligenes sp., Rhizobium etli, Paracoccus denitrificans, Acinetobacter sp., Rhodobacter sphaeroides, Methylobacterium extorquens, Pseudomonas oleovorans, Pseudomonas aeruginosa, Rhodococcus ruber, and Zoogloea ramigera. Dimeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Thiocystis violacea and Chromatium vinosum. Preferably, the polyhydroxyalkanoic acid synthase protein is an Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein, and more preferably, the Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein encoded by the Alcaligenes eutrophus phaC polyhydroxyalkanoic acid synthase structural gene. The acyl-CoA synthetase protein may be any acyl-CoA synthetase protein suitable for the preparation of polyester materials according to the inventive processes. Sources of acyl-CoA synthetase proteins include, but are not limited to, Alcaligenes eutrophus, Methanothrix soehngenii, and Aspergillus nidulans. Preferably, the acyl-CoA synthetase protein is a thiokinase protein, and more preferably, a 4-hydroxybutyrate thiokinase protein.
The invention discloses methods for the preparation of polyester materials. The polyester materials may be any polyester material obtained via the inventive processes. The polyester may be a homopolyester or a copolyester. Preferably, the homopolyester is poly(4-hydroxybutyric acid) or poly(3-hydroxybutyric acid). The copolyester is preferably poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid). In a preferred embodiment, the method comprises the steps of a) obtaining a cell containing a nucleic acid segment, the nucleic acid segment encoding a polyhydroxyalkanoic acid synthase protein and encoding a fatty acid:acyl-CoA transferase protein; b) establishing a culture of the cell; c) culturing the cell under conditions suitable for the production of a polyester; and d) isolating the polyester from the cell. The polyhydroxyalkanoic acid synthase protein may generally be any polyhydroxyalkanoic acid synthase protein suitable for the production of polyester materials according to the inventive method. The polyhydroxyalkanoic acid synthase protein may be monomeric or dimeric. Monomeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Rhizobium meliloti, Alcaligenes eutrophus, Alcaligenes sp., Rhizobium etli, Paracoccus denitrificans, Acinelobacter sp., Rhodobacter sphaeroides, Methylobacterium extorquens, Pseudomonas oleovorans, Pseudomonas aeruginosa, Rhodococcus ruber, and Zoogloea ramigera. Dimeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Thiocystis violacea and Chromatium vinosum. Preferably, the polyhydroxyalkanoic acid synthase protein is an Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein, and more preferably, the Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein encoded by the Alcaligenes eutrophus phaC polyhydroxyalkanoic acid synthase structural gene. The fatty acid:acyl-CoA transferase protein may be any fatty acid:acyl-CoA transferase protein suitable for the preparation of polyester materials according to the inventive method. Sources of acyl-CoA synthetase proteins include, but are not limited to, Alcaligenes eutrophus, Methanothrix soehngenii, and Aspergillus nidulans. Preferably, the fatty acid:acyl-CoA transferase protein is a 4-hydroxybutyrate:acyl-CoA transferase protein, more preferably a Clostridium kluyveri 4-hydroxybutyrate:acyl-CoA transferase protein, and most preferably, the Clostridium kluyveri 4-hydroxybutyrate:acyl-CoA transferase protein encoded by the Clostridium kluyveri orfZ 4-hydroxybutyrate:acyl-CoA transferase structural gene. The culture may contain glucose or any material capable of conversion to glucose by the cell, as a carbon source. The culture may contain 4-hydroxybutyric acid, the sodium salt of 4-hydroxybutyric acid, xcex3-butyrolactone, 1,4-butanediol, 4-hydroxyvaleric acid, xcex3-valerolactone, 1,4-pentanediol, 3-hydroxybutyric acid, the sodium salt of 3-hydroxybutyric acid, a hydroxypropionic acid, a hydroxybutyric acid, a hydroxyvaleric acid, a hydroxycaproic acid, a hydroxyheptanoic acid, a hydroxyoctanoic acid, a hydroxydecanoic acid, xcex3-caprolactone, xcex3-heptanoloactone, xcex3-octanolactone, xcex3-decanolactone, or any material capable of conversion to 4-hydroxybutyric acid by the cell. The culture may contain molecular oxygen. Molecular oxygen may be present due to bubbling of oxygen gas, or an oxygen containing gas, such as air, through the culture. Alternatively, the molecular oxygen may be present due to agitation of the culture. The cell may contain an protein capable of hydrolyzing lactones to the corresponding hydroxyalkanoic acids. Such proteins may include, but are not limited to, the 1,4-lactonase proteins from rat and humans. To facilitate conversion of 2-oxoglutarate to polyester materials, the cell may further comprise a 2-oxoglutarate decarboxylase protein (for example, from Leuconostoc oenos or Euglena gracilis) and a 4-hydroxybutyrate dehydrogenase protein (for example, from C. Kluyveri or A. eutrophus). To facilitate conversion of succinate to polyester materials, the cell may further comprise a succinate:acetyl-CoA transferase protein (for example, from C. kluyveri), a succinate-semialdehyde dehydrogenase protein (for example, from C. kluyveri), and a 4-hydroxybutyrate dehydrogenase protein. To facilitate conversion of succinyl-CoA to polyester materials, the cell may further comprise a succinate-semialdehyde dehydrogenase protein, and a 4-hydroxybutyrate dehydrogenase protein. To facilitate conversion of propionyl-CoA to polyester materials, the cell may further comprise a 2-methylcitrate synthase protein (for example, from Saccharomyces cerevisiae), a 2-methylcitrate dehyratase protein (for example, from Saccharomyces cerevisiae), a 2-methylisocitrate dehydratase protein (for example, from Saccharomyces cerevisiae), a 2-methylisocitrate lyase protein (for example, from Saccharomyces cerevisiae), a succinate:acetyl-CoA transferase protein (for example, from C. kluyveri), a succinate-semialdehyde dehydrogenase protein, and a 4-hydroxybutyrate dehydrogenase protein. It will be apparent to those of skill in the art that different combinations of polyhydroxyalkanoic acid synthases, fatty acid:acyl-CoA transferases, and chemical substrates can be used according to the inventive methods to afford polyester materials.
The invention further describes alternative methods for the preparation of polyester materials. In a preferred embodiment, the method comprises the steps of a) obtaining a cell containing a nucleic acid segment, the nucleic acid segment encoding a polyhydroxyalkanoic acid synthase protein and encoding an acyl-CoA synthetase protein; b) establishing a culture of the cell; c) culturing the cell under conditions suitable for the production of a polyester; and d) isolating the polyester from the cell. The polyester materials may be any polyester material obtained via the inventive processes. The polyester may be a homopolyester or a copolyester. Preferably, the homopolyester is poly(4-hydroxybutyric acid) or poly(3-hydroxybutyric acid). The copolyester is preferably poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid). The polyhydroxyalkanoic acid synthase protein may generally be any polyhydroxyalkanoic acid synthase protein suitable for the production of polyester materials according to the inventive method. The polyhydroxyalkanoic acid synthase protein may be monomeric or dimeric. Monomeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Rhizobium meliloti, Alcaligenes eutrophus, Alcaligenes sp., Rhizobium etli, Paracoccus denitrificans, Acinetobacter sp., Rhodobacter sphaeroides, Methylobacterium extorquens, Pseudomonas oleovorans, Pseudomonas aeruginosa, Rhodococcus ruber, and Zoogloea ramigera. Dimeric polyhydroxyalkanoic acid synthase proteins include, but are not limited to, those from Thiocystis violacea and Chromatium vinosum. Preferably, the polyhydroxyalkanoic acid synthase protein is an Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein, and more preferably, the Alcaligenes eutrophus polyhydroxyalkanoic acid synthase protein encoded by the Alcaligenes eutrophus phaC polyhydroxyalkanoic acid synthase structural gene. The acyl-CoA synthetase protein may be any acyl-CoA synthetase protein suitable for the preparation of polyester materials according to the inventive processes. Sources of acyl-CoA synthetase proteins include, but are not limited to, Alcaligenes eutrophus, Methanothrix soehngenii, and Aspergillus nidulans. Preferably, the acyl-CoA synthetase protein is a thiokinase protein, and more preferably, a 4-hydroxybutyrate thiokinase protein. The culture may contain glucose or any material capable of conversion to glucose by the cell, as a carbon source. The culture may contain 4-hydroxybutyric acid, the sodium salt of 4-hydroxybutyric acid, xcex3-butyrolactone, 1,4-butanediol, 4-hydroxyvaleric acid, xcex3-valerolactone, 1,4-pentanediol, 3-hydroxybutyric acid, the sodium salt of 3-hydroxybutyric acid, a hydroxypropionic acid, a hydroxybutyric acid, a hydroxyvaleric acid, a hydroxycaproic acid, a hydroxyheptanoic acid, a hydroxyoctanoic acid, a hydroxydecanoic acid, xcex3-caprolactone, xcex3-heptanoloactone, xcex3-octanolactone, xcex3-decanolactone, or any material capable of conversion to 4-hydroxybutyric acid by the cell. The culture may contain molecular oxygen. Molecular oxygen may be present due to bubbling of oxygen gas, or an oxygen containing gas, such as air, through the culture. Alternatively, the molecular oxygen may be present due to agitation of the culture. The cell may contain an enzyme capable of hydrolyzing lactones to the corresponding hydroxyalkanoic acids. Such proteins may include, but are not limited to, the 1,4-lactonase proteins from rat and humans. To facilitate conversion of 2-oxoglutarate to polyester materials, the cell may further comprise a 2-oxoglutarate decarboxylase protein and a 4-hydroxybutyrate dehydrogenase protein. To facilitate conversion of succinate to polyester materials, the cell may further comprise a succinate:acetyl-CoA transferase protein, a succinate-semialdehyde dehydrogenase protein, and a 4-hydroxybutyrate dehydrogenase protein. To facilitate conversion of succinyl-CoA to polyester materials, the cell may further comprise a succinate-semialdehyde dehydrogenase protein, and a 4-hydroxybutyrate dehydrogenase protein. To facilitate conversion of propionyl-CoA to polyester materials, the cell may further comprise a 2-methylcitrate synthase protein, a 2-methylcitrate dehyratase protein, a 2-methylisocitrate dehydratase protein, a 2-methylisocitrate lyase protein, a succinate:acetyl-CoA transferase protein, a succinate-semialdehyde dehydrogenase protein, and a 4-hydroxybutyrate dehydrogenase protein. It will be apparent to those of skill in the art that different combinations of polyhydroxyalkanoic acid synthases, acyl-CoA synthetases, and chemical substrates can be used according to the inventive methods to afford polyester materials.
In a further alternative embodiment, the preparation of polyester materials may be achieved by the co-expression of a polyhydroxyalkanoic acid synthase protein, an acyl kinase protein, and a phosphotransacylase protein. A source of acyl kinase includes, but is not limited to, the butyrate kinase from Clostridium acetobutylicum. A source of a phosphotransacylase protein includes, but is not limited to, the phosphotransbutyrylase protein from Clostridium acetobutylicum. 
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.